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The effect of silver ions on the respiratory chain of
Escherichia
colil
P.
D.
BRAGG
ND
D.
J. RAINNIE
Deparltnetil of Biochetlzistty, Utiiversity of'Britislz Colrrt~~bia,arrcoriver, Britislr ColrrtnbiaAccepted February 18, 1974BKAGG,
.
D.,
and
D.
J.
RAINNIE.974. The effect of silver ions on the respiratory chain ofEsclrerichia coli. Can.
J.
Microbiol.
20:
883-889.Silver ions inhibited the oxidation of glucose, glycerol, fumarate, succinate,
D-
and L-lactate,and endogenous substrates by intact cell suspensions of Esclrerichia coli. Silver ions reactedwith the respiratory chain at two levels. The site nlost sensitive to inhibition was located be-tween the 6-cytochromes and cytochrome
0,.
The second level of inhibition was in the NADHand succinate dehydrogenase regions of the
respiratory
chain, and was situated on thc substrateside of the flavin components.BKAGG,
.D.,
et
D. J.
RAINNIE.974. The erect of silver ions on the respiratory chain ofEsclrericl~ia oli. Can.
J.
Microbiol.
20:
883-889.Les ions d'argent inhibent I'oxidation du glucose, du glycerol, du fumarate, du succinate, du
D-
et L-lactate et Ies substrats endogenes par des suspensions d'Echerichia coli. Les ions d'argentreagissent avec la chaine respiratoire
a
deux niveaux. Le point le plus sensible
B
I'inhibitionest localise entre les cytochromes-b et le cytochrome a2. Le deuxien~e iveau d'inhibition estdans la region de la chaine respiratoire de la dehydrogenase succinate et NADH, et est situesur le c6te du substrat des composants flavines.[Traduit parIe journal]
Introduction
Althou~hsilver ions are known to inhibitI11 previous publications (13, 14) we notedthat, after an initial brief phase of stimulation,the respiratioil of
Eschericlzia coli
metabolizingglucose was inhibited by silver ions. This agreedwith the work of Yudkiil (17) who, some yearsago, found that 10 pM silver sulfate inhibited by50% the reduction of methylene blue by
E.
coli
cells oxidizing glucose, succinate, or lactate.Yudkin also observed that with glucose, dyereduction was initially stimulated by the metalions before inhibition occurred.Chappell and Greville (4) found that lowconcentrations (5-10 pM) of AgNO, stimulatedboth the respiration and adenosinetriphospl~atase(ATPase) activity of rabbit brain mitochondria,and Brierley (3) reported that silver ions inhibitedoxidative phosphbrylation in beef heart mito-chondria. Therefore, it was considered possiblethat this latter process might have been affectedin
E.
coli,
and that the initial stimulatory phase indye reduction and glucose oxidation might re-present an uncoupling action of the silver ions.We subsequently confirmed that hypothesis by
-
transport of succinate into membrane vesicles of
E.
coli
(I 5), there is no illformation on their actiondirectly on the respiratory chain of this or othermicroorganisms. There is some indirect evidenceas to possible sites of action suggested by experi-ments with other metal ions. Thus, in heart muscleparticles zinc ions inhibited at two sites, onebetween cytochromes b and
c,,
and the other atthe flavoprotein level (12). Kleiner and vonJagow (11) using rat liver mitochondria re-examined the site of inhibition of zinc ions andfound that they inhibited the respiratory chain ata site between ubiquinone and cytochrome b. In
E.
coli
respiratory particles zinc ions inhibited thesuccinate oxidase system at the level of succinatedehydrogenase (7).In the present paper we show that silver ionsinhibit the respiratory chain of
E.
coli.
The mostsensitive site of inhibition was between the b-cytochromes and cytochrome
a,.
A further site ofinhibition was located between the site of sub-strate interaction with the respiratory chain andflavoprotein.correlating the magnitude of respiratory stimu-lation by silver ions with the extent of energy
Materials and Methods
coupling in the cells (14). However, the site(s) of
CIzemicaIs
the inhibitory action of silver ions on substrate
Chloride-free glycylglycine was obtained from Nutri-
oxidation
was
not determined either by yudl;in
tional Biochemicals Corporation. Lithium
D-
and
L-
lactate,
3-phosphoglyceraldehyde,
and nicotinamide
or by us.
adenine dinucleotide (NAD+) were purchased fromCalbiochem. Silver nitrate was Fisher certified ACS'Received October 4, 1973. grade.
Tris(hydroxymethyl)aminomethane
(Tris) (free
 
884
CAN.
J.
MICROBIOL. VOL.
20,
1974
base) and ammonium sulfate were of ultrapure-gradeobtained from Schwarz-Mann Division of Becton,Dickinson and Company. All other chemicals werereagent-grade.
Cultrrre Conditions and Preparation of Cell Susper~sior~sEscherichia coli
strain 482 of the culture collection ofthe National Research Council of Canada was used in allinvestigations reported here. The cells were cultured intwo ways as described below.
Culture Method A
The cells were grown on a minimal salts medium ofthe following composition: 0.7% K2HP04; 0.3%KH2P04;0.1% (NH4),S04, and 0.02% MgS04. Carbonsources and the concentrations used were glucose 0.4%,glycerol 0.4%,
DL-~C~~C
cid 0.8%, sodium succinate0.4%, and potassium fumarate 0.4%. The medium wasadjusted to pH 7.0 before autoclaving. Growth wasinitiated by inoculating 600 ml of sterile medium with a10% (vlv) overnight culture grown on the appropriatecarbon source. The culture was vigorously aerated via asintered glass sparger (Kimax 12C) at an air flow rate of6.7 literslmin. Growth at 37' was followed by measuringthe absorbance at 420 nm of an aliquot of the culture ina cuvette with 1 cm path length. The cells were harvestedin the late exponential phase of growth by centrifugationat 5900
x
g
for 10 min at 4". The cells were washed twiceby centrifugation from 0.85% NaCl at 4300
x
g
for 10min and were resuspended at a dilution of 1
:
10 (wlv) inthe buffer indicated.
Culture Method
B
The cells were grown on the minimal medium used inmethod
A
but with 0.2% (NH4),S04 and with theaddition of
6
pM ferric citrate. The carbon source was1.4% (w/v) sodium succinate.6H20. Growth was ini-tiated by inoculating 350 ml of the sterile medium in a1-liter erlenmeyer flask with a 10% (v/v) culture grownfor 9 h on the same medium. The culture was vigorouslyshaken (120 strokeslmin) at 37' for 15 h on a reciprocatingshaker (New Brunswick Scientific Co.). The cells wereharvested by centrifugation at 4400
x
g
for 15 min;washed with 0.01 M Tris-HCI buffer, pH 7.3, containing0.01 M MgC1, (350 ml), and then withO.lMglycylglycinebuffer, pH 8.0 (40 ml); and recovered by centrifugation.For experiments with whole cells the final washingstep with glycylglycine buffer was repeated, and the cellssuspended in a volume of the same buffer equivalent to15 times the wet weight of the cells. The cell suspensionwas kept at room temperature during the duration (about
3
h) of the experiment.For the preparation of respiratory particles the wholecells were suspended in a volume of glycylglycine bufferequivalent to 10 times the wet weight of the cells.
Preparation of Respiratory Particles
A few crystals of deoxyribonuclease (DNase) (Worth-ington Biochemical Corp.) and M MgCI, (final concen-tration, 4mM) were added to the cell suspension whichwas then disrupted at
O"
n a French pressure cell (Aminco)operated at 20 000 psi. The extract was centrifuged at27000
x
g
for 15 min. The supernatant fraction wasrecentrifuged at 176 000
x
g
for 2
h
and the well-packedpellet of respiratory particles suspended in a volume of0.1 M glycylglycine buffer, pH 8.0, equivalent to 5 timesthe wet weight of cells from which the particles werederived. The suspension was stored at 0" for the durationof the experiment.
Assays
Oxygen uptake was measured with an oxygen monitor(Yellow Springs Instrument Co. model 55) equipped witha Clark type oxygen electrode (Yellow Springs Instru-ment Co. model 5533) and a linear chart recorder.Reduced minus oxidized difference spectra were recordedusing a Cary mode! 15 spectrophotometer equipped witha 0.1-absorbance slide-wire. The redox state of a respira-tory chain component was measured by comparing theabsorbance increment caused by the reduced componentat a selected wave!ength in the reduced minus oxidizeddifference spectrum to the absorbance increment obtainedin the presence of sodium dithionite which fully reducedthe components of the respiratory chain. The selectedwavelengths for flavoprotein and cytochromes
b,,
a,,
and
a2
were 465 nm, 559 nm, 590 nm, and 628 nm.respectively. The components in the reference cuvettewere maintained in the oxidized form with 0.05%
H202.
Results
EfSect of Silver Ions on Substrate Oxidation
The effect of
86
pM
AgNO, on the oxidationof glucose, glycerol, fumarate, and D-lactate bywhole cells is shown in Fig. 1. Inhibition ofoxygen uptake occurred rapidly with all of thesesubstrates after the addition of AgNO,. That theinhibition was due to silver ions was shown incontrol experiments where no inhibition wasobtained by addition of KNO,. The effect ofsilver ions on the oxidation of succinate andL-lactate was similar to that on fumarate andD-lactate. The cells used in these experimentswere grown on the same substrate as was sub-sequently used in the oxygen-uptake experiment.However, the same pattern of results were ob-tained if the effect of silver ions on substrateoxidation was measured using cells grown ondifferent carbon sources.To avoid the uncertainties introduced by apossible effect of silver ions on the transport ofsubstrate, the effect of this inhibitor on the oxida-tion of endogenous substrates was examined.This experiment was carried out in a different wayto those shown in Fig. 1. An aliquot of a cellsuspension was added to buffer containingAgNO, and the uptake of oxygen then measured.Even in the absence of inhibitor nonlinear ratesof oxygen uptake were obtained. For this reasonrates were measured at two particular time in-tervals
(4
and
8
min) after addition of AgNO, tothe cell suspension. The rates were then compared
 
BRAGGANDRAINNIE:
EFFECT
OF
AgNO,
ON
E.COLI
MINUTES
FIG. 1. Effect of silver ions on oxidation of substrates by intact cells of
E.
coli.
Oxygen uptake was measuredat 37" in suspensions of cells grown (culture method A) on, and assayed with the same carbon source. Theassay system
(5.1
ml) contained 288 mM glycylglycine-KOH bufier, pH 7.0, and 0.4 mM KCI. At the indi-cated time (top of figure) 0.05 ml of cell suspension (C), 0.05 ml M substrate
(S),
and 0.03 ml 15 mM AgNO,(Ag) were added. In the control (broken line) only the initial addition of cell suspension and substrate weremade. Oxygen level is expressed as
%
saturation.
with those obtained at these two time intervals inTo rule out a possible action on other metabolicthe absence of inhibitor to give
"%
initial value." pathways the effect of AgNO, on the succinateAs shown in Fig. 2A oxidation of endogenousand reduced NAD (NADH) oxidase activity ofsubstrates was inhibited by silver ions and the respiratory particles was examined (Fig. 2B).extent of inhibition increased with time. The particles were preincubated for
5
min with
FIG. 2.Effect of concentration of AgNO, on the rate of oxidation of endogenous substrates by wholecells (A), and on the oxidation of NADH and succinate by respiratory particles
(B).
In A the oxygen con-sumption was measured at 37" following the addition of 1.0 ml cell suspension to 3.5 ml 0.1
M
glycylglyci?ebuffer, pH
8.0,
containing the indicated concentration of AgNO,. The rates of oxygen uptake at
4
and 8 mlnafter the cell suspension was added to the AgNOa are plotted. Initial values for rate of oxygen uptake in theabsence of inhibitor:
4
min, 2.45 pg atoms O/min per gram cells; 8 min, 1.93 pg atoms O/min per gram cells.In
B
1.5 ml respiratory particle suspension
(6.2
mg protein) was preincubated at 21' for 5 min with AgNO,.NADH (2.8 prnol) or disodium succinate
(5
pmol) was then added and the rate of oxygen uptake measufedby the spectrophotometric technique (Ref.
1).
Initial value: NADH oxidase, 0.37 pg atoms O/mm per mllll-gram protein; succinate oxidase, 0.15 pg atoms O/min per milligram protein.
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